scholarly journals Different Classes of Antibiotics Differentially Influence Shiga Toxin Production

2010 ◽  
Vol 54 (9) ◽  
pp. 3790-3798 ◽  
Author(s):  
Colleen Marie McGannon ◽  
Cynthia Ann Fuller ◽  
Alison Ann Weiss
Keyword(s):  
Shiga Toxin ◽  
Vero Cells ◽  
Toxin Production ◽  
Lytic Cycle ◽  
Temperate Bacteriophage ◽  
E Coli ◽  
Growth Inhibitory ◽  
Target Dna ◽  
Stress Signals ◽  
Very High

ABSTRACTShiga toxin (Stx) inEscherichia coliO157:H7 is encoded as a late gene product by temperate bacteriophage integrated into the chromosome. Phage late genes, includingstx, are silent in the lysogenic state. However, stress signals, including some induced by antibiotics, trigger the phage to enter the lytic cycle, and phage replication and Stx production occur concurrently. In addition to the Stx produced by O157:H7, phage produced by O157:H7 can infect harmless intestinalE. coliand recruit them to produce Shiga toxin. To understand how antibiotics influence Stx production, Stx lysogens were treated with different classes of antibiotics in the presence or absence of phage-sensitiveE. coli, and Stx-mediated inhibition of protein synthesis was monitored using luciferase-expressing Vero cells. Growth-inhibitory levels of antibiotics suppressed Stx production. Subinhibitory levels of antibiotics that target DNA synthesis, including ciprofloxacin (CIP) and trimethoprim-sulfamethoxazole, increased Stx production, while antibiotics that target the cell wall, transcription, or translation did not. More Stx was produced whenE. coliO157:H7 was incubated in the presence of phage-sensitiveE. colithan when grown as a pure culture. Remarkably, very high levels of Stx were detected even when growth of O157:H7 was completely suppressed by CIP. In contrast, azithromycin significantly reduced Stx levels even when O157:H7 viability remained high.

scholarly journals The CI Repressors of Shiga Toxin-Converting Prophages Are Involved in Coinfection of Escherichia coli Strains, Which Causes a Down Regulation in the Production of Shiga Toxin 2

2008 ◽  
Vol 190 (13) ◽  
pp. 4722-4735 ◽  
Author(s):  
R. Serra-Moreno ◽  
J. Jofre ◽  
M. Muniesa
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Antibiotic Resistance Gene ◽  
Vero Cells ◽  
Toxin Production ◽  
Lytic Cycle ◽  
Host Chromosome ◽  
E Coli ◽  
Shiga Toxin 2 ◽  
K 12

ABSTRACT Shiga toxins (Stx) are the main virulence factors associated with a form of Escherichia coli known as Shiga toxin-producing E. coli (STEC). They are encoded in temperate lambdoid phages located on the chromosome of STEC. STEC strains can carry more than one prophage. Consequently, toxin and phage production might be influenced by the presence of more than one Stx prophage on the bacterial chromosome. To examine the effect of the number of prophages on Stx production, we produced E. coli K-12 strains carrying either one Stx2 prophage or two different Stx2 prophages. We used recombinant phages in which an antibiotic resistance gene (aph, cat, or tet) was incorporated in the middle of the Shiga toxin operon. Shiga toxin was quantified by immunoassay and by cytotoxicity assay on Vero cells (50% cytotoxic dose). When two prophages were inserted in the host chromosome, Shiga toxin production and the rate of lytic cycle activation fell. The cI repressor seems to be involved in incorporation of the second prophage. Incorporation and establishment of the lysogenic state of the two prophages, which lowers toxin production, could be regulated by the CI repressors of both prophages operating in trans. Although the sequences of the cI genes of the phages studied differed, the CI protein conformation was conserved. Results indicate that the presence of more than one prophage in the host chromosome could be regarded as a mechanism to allow genetic retention in the cell, by reducing the activation of lytic cycle and hence the pathogenicity of the strains.

scholarly journals Nonpathogenic Escherichia coli Can Contribute to the Production of Shiga Toxin

2003 ◽  
Vol 71 (6) ◽  
pp. 3107-3115 ◽  
Author(s):  
Shantini D. Gamage ◽  
Jane E. Strasser ◽  
Claudia L. Chalk ◽  
Alison A. Weiss
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Gastrointestinal Disease ◽  
Intestinal Flora ◽  
Gene Promoter ◽  
Toxin Production ◽  
Lytic Cycle ◽  
Resistant Bacteria ◽  
E Coli ◽  
Shiga Toxin 2

ABSTRACT The food-borne pathogen, Escherichia coli O157:H7, has been associated with gastrointestinal disease and the life-threatening sequela hemolytic uremic syndrome. The genes for the virulence factor, Shiga toxin 2 (Stx2), in E. coli O157:H7 are encoded on a temperate bacteriophage under the regulation of the late gene promoter. Induction of the phage lytic cycle is required for toxin synthesis and release. We investigated the hypothesis that nonpathogenic E. coli could amplify Stx2 production if infected with the toxin-encoding phage. Toxin-encoding phage were incubated with E. coli that were either susceptible or resistant to the phage. The addition of phage to phage-susceptible bacteria resulted in up to 40-fold more toxin than a pure culture of lysogens, whereas the addition of phage to phage-resistant bacteria resulted in significantly reduced levels of toxin. Intestinal E. coli isolates incubated with Shiga toxin-encoding phage produced variable amounts of toxin. Of 37 isolates, 3 produced significantly more toxin than was present in the inoculum, and 1 fecal isolate appeared to inactivate the toxin. Toxin production in the intestine was assessed in a murine model. Fecal toxin recovery was significantly reduced when phage-resistant E. coli was present. These results suggest that the susceptibility of the intestinal flora to the Shiga toxin phage could exert either a protective or an antagonistic influence on the severity of disease by pathogens with phage-encoded Shiga toxin. Toxin production by intestinal flora may represent a novel strategy of pathogenesis.

Influence of Cultural Conditions on Cytotoxin Production by Salmonella enteritidis

1992 ◽  
Vol 55 (1) ◽  
pp. 28-33 ◽  
Author(s):  
RATIH DEWANTI ◽  
MICHAEL P. DOYLE
Keyword(s):  
Shiga Toxin ◽  
Salmonella Enteritidis ◽  
Incubation Temperature ◽  
Vero Cells ◽  
Toxin Production ◽  
Growth Media ◽  
Optimal Conditions ◽  
Early Stationary Phase ◽  
Cultural Conditions ◽  
Optimal Ph

The ability of Salmonella enteritidis strain 11013 to produce cytotoxic activity against Vero cells was determined under different cultural conditions. The toxin, which was not neutralizable with antiserum to Shiga toxin or Escherichia coli verotoxin-1 or verotoxin-2, was principally cell-associated and was produced primarily during the early stationary phase of growth. Trypticase soy broth was the best of three media evaluated for toxin production. Bacteria produced toxin in the range of pH 4.5 to 8.0 and at 12 to 42°C, with the optimal pH and temperature for toxin production at pH 7.0 and 37°C, respectively. Release of cellular cytotoxin into growth media was induced by growing salmonellae at extremes of pH (4.5 or 8.0) or at high incubation temperature (42°C). The Vero cell CD50 of S. enteritidis lysates of cells grown under optimal conditions was a titer of 150 ± 50 per mg of lysate protein. Although the significance of ingesting preformed Salmonella cytotoxin in human disease is unknown, it can be implied from these results that toxin would not be produced in foods held refrigerated at ≤7°C or acidified at ≤pH 4.0.

scholarly journals Replication of plasmids derived from Shiga toxin-converting bacteriophages in starved Escherichia coli

Microbiology ◽  
2011 ◽  
Vol 157 (1) ◽  
pp. 220-233 ◽  
Author(s):  
Bożena Nejman ◽  
Beata Nadratowska-Wesołowska ◽  
Agnieszka Szalewska-Pałasz ◽  
Alicja Węgrzyn ◽  
Grzegorz Węgrzyn
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Transcriptional Activation ◽  
Stringent Response ◽  
Toxin Production ◽  
Replication Initiation ◽  
Host Cells ◽  
Regulation Of Transcription ◽  
E Coli ◽  
Dna Replication Initiation

The pathogenicity of Shiga toxin-producing Escherichia coli (STEC) depends on the expression of stx genes that are located on lambdoid prophages. Effective toxin production occurs only after prophage induction, and one may presume that replication of the phage genome is important for an increase in the dosage of stx genes, positively influencing their expression. We investigated the replication of plasmids derived from Shiga toxin (Stx)-converting bacteriophages in starved E. coli cells, as starvation conditions may be common in the intestine of infected humans. We found that, unlike plasmids derived from bacteriophage λ, the Shiga toxin phage-derived replicons did not replicate in amino acid-starved relA + and relA − cells (showing the stringent and relaxed responses to starvation, respectively). The presence of the stable fraction of the replication initiator O protein was detected in all tested replicons. However, while ppGpp, the stringent response effector, inhibited the activities of the λ P R promoter and its homologues from Shiga toxin-converting bacteriophages, these promoters, except for λ P R, were only weakly stimulated by the DksA protein. We suggest that this less efficient (relative to λ) positive regulation of transcription responsible for transcriptional activation of the origin contributes to the inhibition of DNA replication initiation of Shiga toxin-converting bacteriophages in starved host cells, even in the absence of ppGpp (as in starved relA − hosts). Possible clinical implications of these results are discussed.

scholarly journals Contribution of the Twin Arginine Translocation System to the Virulence of Enterohemorrhagic Escherichia coli O157:H7

2003 ◽  
Vol 71 (9) ◽  
pp. 4908-4916 ◽  
Author(s):  
Nathalie Pradel ◽  
Changyun Ye ◽  
Valérie Livrelli ◽  
Jianguo Xu ◽  
Bernard Joly ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Immunoblot Analysis ◽  
Vero Cells ◽  
Soft Agar ◽  
Enterohemorrhagic Escherichia Coli ◽  
E Coli ◽  
Twin Arginine Translocation ◽  
Tat System ◽  
K 12

ABSTRACT Shiga toxin-producing Escherichia coli O157:H7 is a major food-borne infectious pathogen. In order to analyze the contribution of the twin arginine translocation (TAT) system to the virulence of E. coli O157:H7, we deleted the tatABC genes of the O157:H7 EDL933 reference strain. The mutant displayed attenuated toxicity on Vero cells and completely lost motility on soft agar plates. Further analyses revealed that the ΔtatABC mutation impaired the secretion of the Shiga toxin 1 (Stx1) and abolished the synthesis of H7 flagellin, which are two major known virulence factors of enterohemorrhagic E. coli O157:H7. Expression of the EDL933 stxAB 1 genes in E. coli K-12 conferred verotoxicity on this nonpathogenic strain. Remarkably, cytotoxicity assay and immunoblot analysis showed, for the first time, an accumulation of the holotoxin complex in the periplasm of the wild-type strain and that a much smaller amount of StxA1 and reduced verotoxicity were detected in the ΔtatC mutant cells. Together, these results establish that the TAT system of E. coli O157:H7 is an important virulence determinant of this enterohemorrhagic pathogen.

scholarly journals Characterization of a Shiga Toxin-Encoding Temperate Bacteriophage of Shigella sonnei

2001 ◽  
Vol 69 (12) ◽  
pp. 7588-7595 ◽  
Author(s):  
Eckhard Strauch ◽  
Rudi Lurz ◽  
Lothar Beutin
Keyword(s):  
Shiga Toxin ◽  
Regulatory Region ◽  
Shigella Sonnei ◽  
Shigella Dysenteriae ◽  
Nucleotide Sequencing ◽  
Temperate Bacteriophage ◽  
Cross Hybridization ◽  
E Coli ◽  
K 12

ABSTRACT A Shiga toxin (Stx)-encoding temperate bacteriophage ofShigella sonnei strain CB7888 was investigated for its morphology, DNA similarity, host range, and lysogenization inShigella and Escherichia coli strains. Phage 7888 formed plaques on a broad spectrum of Shigella strains belonging to different species and serotypes, including Stx-producingShigella dysenteriae type 1. With E. coli, only strains with rough lipopolysaccharide were sensitive to this phage. The phage integrated into the genome of nontoxigenic S. sonneiand laboratory E. coli K-12 strains, which became Stx positive upon lysogenization. Moreover, phage 7888 is capable of transducing chromosomal genes in E. coli K-12. The relationships of phage 7888 with the E. coli Stx1-producing phage H-19B and the E. coli Stx2-producing phage 933W were investigated by DNA cross-hybridization of phage genomes and by nucleotide sequencing of an 8,053-bp DNA region of the phage 7888 genome flanking the stx genes. By these methods, a high similarity was found between phages 7888 and 933W. Much less similarity was found between phages H-19B and 7888. As in the other Stx phages, a regulatory region involved in Q-dependent expression is found upstream of stxA and stxB (stx gene) in phage 7888. The morphology of phage 7888 was similar to that of phage 933W, which shows a hexagonal head and a short tail. Our findings demonstrate that stx genes are naturally transferable and are expressed in strains of S. sonnei, which points to the continuous evolution of human-pathogenic Shigella by horizontal gene transfer.

scholarly journals A putative microcin amplifies Shiga toxin 2a production ofEscherichia coliO157:H7

2019 ◽  
Author(s):  
Hillary M. Figler ◽  
Lingzi Xiaoli ◽  
Kakolie Banerjee ◽  
Maria Hoffmann ◽  
Kuan Yao ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Bacterial Infections ◽  
Bacterial Species ◽  
Treatment Options ◽  
Gene Clusters ◽  
Toxin Production ◽  
Additional Treatment ◽  
Proteinase K ◽  
Gut Microflora ◽  
E Coli

AbstractEscherichia coliO157:H7 is a foodborne pathogen, implicated in various multi-state outbreaks. It encodes Shiga toxin on a prophage, and Shiga toxin production is linked to phage induction. AnE. colistrain, designated 0.1229, was identified that amplified Stx2a production when co-cultured withE. coliO157:H7 strain PA2. Growth of PA2 in 0.1229 cell-free supernatants had a similar effect, even when supernatants were heated to 100°C for 10 min, but not after treatment with Proteinase K. The secreted molecule was shown to use TolC for export and the TonB system for import. The genes sufficient for production of this molecule were localized to a 5.2 kb region of a 12.8 kb plasmid. This region was annotated, identifying hypothetical proteins, a predicted ABC transporter, and a cupin superfamily protein. These genes were identified and shown to be functional in two otherE. colistrains, and bioinformatic analyses identified related gene clusters in similar and distinct bacterial species. These data collectively suggestE. coli0.1229 and otherE. coliproduce a microcin that induces the SOS response in target bacteria. Besides adding to the limited number of microcins known to be produced byE. coli, this study provides an additional mechanism by whichstx2aexpression is increased in response to the gut microflora.ImportanceHow the gut microflora influences the progression of bacterial infections is only beginning to be understood. Antibiotics are counter-indicated forE. coliO157:H7 infections, and therefore treatment options are limited. An increased understanding of how the gut microflora directs O157:H7 virulence gene expression may lead to additional treatment options. This work identifiedE. colithat enhance the production of Shiga toxin by O157:H7, through the secretion of a proposed microcin. This work demonstrates another mechanism by which non-O157E. colistrains may increase Shiga toxin production, and adds to our understanding of microcins, a group of antimicrobials that are less well understood than colicins.

Prevalence and Characteristics of Shiga Toxin-Producing Escherichia coli in Beef Cattle Slaughtered on Prince Edward Island

2000 ◽  
Vol 63 (11) ◽  
pp. 1583-1586 ◽  
Author(s):  
R. DOUGLAS SCHURMAN ◽  
HARRY HARIHARAN ◽  
SUSAN B HEANEY ◽  
KRIS RAHN
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Prince Edward Island ◽  
Toxin Production ◽  
Positive Sample ◽  
Chain Reaction ◽  
Antimicrobial Drugs ◽  
E Coli ◽  
Human Illness ◽  
Polymerase Chain

Fecal swabs obtained from a random sample of 1,000 beef slaughter steers and heifers from 123 Prince Edward Island (P.E.I.) farms were examined for the presence of Shiga toxin-producing Escherichia coli (STEC) using a Vero cell assay (VCA). Multiple isolates from each positive sample were tested similarly. VCA-positive isolates were confirmed as E. coli biochemically, tested for drug resistance, serotyped, and tested by polymerase chain reaction (PCR). Animals were classified as positive when an isolate was positive on VCA and the presence of the gene responsible for toxin production was confirmed by PCR. The prevalence of STEC in beef slaughter steers and heifers on P.E.I. was 4% (40 of 1,000). The total number of isolates was 43, and these comprised 26 serotypes, including 13 isolates belonging to 6 serotypes known to be associated with human illness. The most frequently isolated STEC serotype was E. coli O157 (5 isolates out of 43). Of the five E. coli O157 isolates, four were E. coli O157:H7, a serious human pathogen. The majority of STEC isolates, including all O157:H7, isolates, were susceptible to 16 commonly used antimicrobial drugs. According to PCR, 65% of the STEC isolates had the gene for Stx1. Four of these isolates, including two O157: H7, had genes for Shiga toxin (Stx)1 and Stx2.

Discovery of Enteric Pathogens in the Alaskan Subsistence Diet

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S129-S129
Author(s):  
Grace Leu-Burke ◽  
Robert Beacham ◽  
Courtney Bennetts
Keyword(s):  
Shiga Toxin ◽  
Toxin Production ◽  
Enteric Pathogen ◽  
Enteric Pathogens ◽  
Transmission Risk ◽  
Health Concern ◽  
E Coli ◽  
Wild Game ◽  
And Migration ◽  
Pathogen Colonization

Abstract Objectives Transmission of enteric pathogens from food ingestion is an ongoing public health concern, with commensal bacteria in ruminant animal species causing human disease. Enteric pathogens Salmonella, Shigella, and Shiga toxin producing Escherichia coli (STEC) have been isolated from domesticated animals. However, the Alaskan subsistence diet relies on wild game, such as reindeer, caribou, and moose for their food supply. Research concerning enteric pathogens in wildlife has not established. Therefore, we conducted a pilot survey on moose and reindeer to determine potential enteric pathogen transfer risk. Methods Between July 2018 and January 2019, we collected 72 fecal samples from reindeer and moose migrating in Fairbanks, Anchorage, and the Matanuska Valley. Samples were cultured for enteric pathogens, including E coli 0157, using standard clinical microbial process. Phenotypic Shiga toxin production was verified by enzyme immunoassay. Results Reindeer were statistically significant for enteric pathogens when compared to moose (P < .05) Eighty percent of the reindeer population were colonized for either Shigella, Yersinia, or Shiga toxin-producing E coli, with 20% positive for multiple pathogens. Non-0157 Shiga toxin production was observed in 30% of reindeer samples, generated by a sorbitol fermenting E coli. In contrast, moose population showed a near absence of enteric pathogens with only 5% positive for Shigella. Salmonella was not identified in either animal. Conclusion Reindeer, moose, and caribou meat are prominent in the Alaskan subsistence diet. Although moose had limited enteric pathogen colonization, reindeer were significant for transmission risk, including non-0157 Shiga toxin producing E coli, which has been linked to hemolytic uremic syndrome. Isolation of a non-0157 STEC in wildlife indicates environmental colonization. Because reindeer and caribou are closely linked in diet and migration, Alaska clinical laboratories should screen for enteric pathogens, including non-0157 Shiga toxins.

scholarly journals Genomic Comparisons and Shiga Toxin Production among Escherichia coli O157:H7 Isolates from a Day Care Center Outbreak and Sporadic Cases in Southeastern Wisconsin

1998 ◽  
Vol 36 (3) ◽  
pp. 727-733 ◽  
Author(s):  
S. Gouveia ◽  
M. E. Proctor ◽  
M.-S. Lee ◽  
J. B. Luchansky ◽  
C. W. Kaspar
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Day Care ◽  
Care Center ◽  
Sporadic Case ◽  
Toxin Production ◽  
Geographic Region ◽  
Day Care Center ◽  
E Coli ◽  
Sporadic Cases

Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (CHEF-PFGE) was used to compare Wisconsin isolates ofEscherichia coli O157:H7, including 39 isolates from a 1994 day care center outbreak, 28 isolates from 18 individuals from the surrounding geographic area with sporadic cases occurring during the 3 months before the outbreak, and 3 isolates, collected in 1995, from patients with hemolytic-uremic syndrome (HUS) who were from eastern Wisconsin counties other than those inhabited by the day care center and sporadic-case individuals. The technique of CHEF-PFGE usingXbaI identified seven highly related restriction endonuclease digestion profiles (REDPs) (93 to 98% similarity) among the 39 day care center isolates and nine XbaI REDPs (63 to 93% similarity) among the 28 isolates from sporadic-case individuals, including REDP 33, which was exhibited by both day care and sporadic-case isolates. PFGE analyses of sequential E. coliO157:H7 isolates from symptomatic day care center attendees revealed that the REDPs of 25 isolates from eight patients were indistinguishable whereas the REDPs of 2 of 6 isolates from two patients differed slightly (93 to 95% similarity). The REDPs of the three isolates from 1995 HUS patients were 78 to 83% similar, with REDP 26 being exhibited by one HUS-associated isolate and an isolate from one day care attendee who did not develop HUS. The genes for both Shiga toxins I and II (stx 1 andstx 2, respectively) were detected in all but one isolate (sporadic case), and Shiga toxin production by the day care center isolates was not significantly different from that of the other isolates, including the three HUS-associated isolates. Analyses ofE. coli O157:H7 isolates from both the day care center outbreak and sporadic cases by CHEF-PFGE permitted us to define the REDP variability of an outbreak and geographic region and demonstrated that the day care center outbreak and a HUS case in 1995 were caused byE. coli O157:H7 strains endemic to eastern Wisconsin.

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